Fig. 7: multicolor FLIM imaging allows dissecting different HIV-1 infection steps during entry.

a Quadruple labeled HIV-1 particles were exposed to triple labeled TZM-bl reporter cells. Images showing the total amount of photons for all seven colors and the three detector channels merged and unmixed with pattern matching analysis are also shown. Scale bar 10 μm. b The same example was processed with the Phasor plot. The dotted square denotes the region subject to analysis due to limitations of SimFCS software utilized (see material and methods). The same channels are shown following the same logic as in (a). The two-dimensional Phasor Plots for each channel are shown and the regions for each species are highlighted in blue for the blue channel, green for the Green Channel and red for the three red channels. These regions were previously calculated for each species and highlight specific lifetimes for both viral particles and/or LaminB-mWasabi, Lifeact-mScarlet and Mito-LSSmKate2 expressed in live cells. Scale bar 10 μm. Three independent experiments were performed with the same conditions. c The average lifetime was employed to unmix all seven channels. The first column shows the overall intensity image for each channel. The second column the calculated fraction of fluorescent protein. The third and fourth row the highlighted fraction for each species. The fifth row the corresponding histogram for the fraction of each species and the red line is the cut off line that separates both species. The last two micrographs show the fraction of each species in terms of intensity (number of photons). Scale bar 10 μm.